Major depressive disorder imposes a large burden on society, afflicting an estimated one in five Americans over their lifetime with its hallmark symptoms of anhedonia, low energy, loss of concentration, and sleep and appetite dysregulation. The prevalence of the disease contrasts with the paucity of rapid, effective treatments. Drug therapies or behavioral therapy typically take eight weeks or more to bring symptom relief, and up to half of patients do not respond to initial treatment. Recent studies have shown that a single low dose of ketamine, an NMDA antagonist that increases glutamate signaling in the brain, can bring about symptom relief in just a few hours, even in patients who do not respond to traditional antidepressant therapies. Ketamine's novel antidepressant mechanism has yet to be fully understood, and there is a pressing need for research in this area because ketamine has side effects (including dissociative symptoms) and abuse potential that make it unsuitable for widespread use. Preliminary studies have shown that ketamine's antidepressant effect can be reproduced by stimulation of glutamatergic cells in the infralimbic prefrontal cortex (ilPFC), an area that has been shown to undergo a spike in glutamate release shortly after ketamine administration. This key finding requires spatial, temporal, and cell-type specificity of stimulation, which is attained by injecting a viral vector into the ilPFC that will express channelrhodopsin 2 (ChR2), a light-sensitive protein, only in cells containing CAMKII, a marker for glutamatergic cells. These cells are then stimulated by a laser, at a frequency and duration intended to mimic the time course of systemic ketamine administration, connected directly to a fiberoptic cannula secured to the skull. Aim 1 will investigate the specific ilPFC axonal projections that underlie this effect. Importantly, the viral vector drives expression of ChR2 throughout the cell, even in the distal terminals of axons. To understand which glutamatergic cells within ilPFC are responsible for the antidepressant effect, the cannula will be placed to direct light to one of the regions that receives terminal projections from the ilPFC, which includes the nucleus accumbens (NAc), lateral habenula (lHb), and dorsal raphe (dR), each of which has been suggested by recent research to be involved in mood regulation and antidepressant response. Aim 2 will characterize the effect of the ilPFC projection within each of these areas. Using immunohistochemistry, slices of each brain area will be stained with a c-Fos antibody, a marker of neural activity, shortly after stimulation to assess if the brain region becomes more or less active as a result of stimulation from the ilPFC. Further, the tissue will be co-stained with antibodies for either CAMKII or GAD, a protein found in interneurons, to assess which cell types the ilPFC projections synapse onto. This project will increase understanding of circuits that underlie a rapid antidepressant effect to help point the way to ketamine-like pharmacotherapies that would be safe for widespread clinical use.